High-Resolution NMR Spectroscopy of Membrane Proteins in Aligned Bicelles

Angelis, Anna A. De; Nevzorov, Alexander A.; Park, Sang Ho; Howell, Stanley C.; Mrse, and Anthony A.; Opella, Stanley J.

doi:10.1021/ja045631y

Cited by 104 publications

(198 citation statements)

References 24 publications

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“…These results are directly transferable to 2D SLF studies of 15 N nuclei, where they are typically applied for structural measurements on uniformly 15 N labeled membrane proteins. Furthermore, with a simple modification in the 2D PDLF sequence, we demonstrate that, in addition to 1 H-13 C couplings, remote 13 C-31 P and 1 H-31 P couplings from phospholipids can be measured in bicelles. It should be mentioned here that the 2D PDLF pulse sequence has been used in previous studies to obtain high-resolution SLF spectra of solids by overcoming the difficulties found when multiple protons are coupled with a 13 C nucleus.…”

Section: Introductionmentioning

confidence: 90%

“…The unique feature of WIM and BLEW spinlocking is that they create an isotropic mixing dipolar coupling Hamiltonian, and therefore the corresponding 2D sequences are called HIMSELF. [54][55][56] In a PDLF experiment, where each pair-wise 13 C-1 H spin interaction in a 13 CH n spin system results in a separate doublet, the observed dipolar splitting Δv is related to the coupling constant d simply by Δv = 2kd; k = 0.42 is the scaling factor of the homonuclear decoupling sequence BLEW-48, this value was calibrated using a 2D 1 H/ 13 C HETCOR experiment and closely matched the theoretically predicted value of 0.424. 39 Therefore, the measurement of heteronuclear dipolar couplings is straightforward using this 2D experiment.…”

Section: Two-dimensional 13 C Separated-local-field Experimentsmentioning

confidence: 99%

“…This is because the different 13 C-31 P dipolar doublets overlapped in the 1D 13 C spectrum are well separated in a 2D plane due to the difference in the chemical shifts of protons to which a particular carbon is correlated.…”

Section: C-31 P and 1 H-31 P Slfmentioning

confidence: 99%

“…The 1 H-31 P couplings are easily recognized in the g 2 and α signals, which appear as tilted doublets. This tilt arises from simultaneous splittings by 13 C-31 P and 1 H-31 P interactions along the horizontal (direct dimension) and vertical (indirect dimension) axes, respectively. Similar splittings for the g 3 and β sites were assigned by comparison to the peaks in the PDLF spectrum.…”

Section: C-31 P and 1 H-31 P Slfmentioning

confidence: 99%

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High-Resolution 2D NMR Spectroscopy of Bicelles To Measure the Membrane Interaction of Ligands

et al. 2007

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Magnetically aligned bicelles are increasingly being used as model membranes in solutionand solidstate NMR studies of the structure, dynamics, topology, and interaction of membraneassociated peptides and proteins. These studies commonly utilize the PISEMA pulse sequence to measure dipolar coupling and chemical shift, the two key parameters used in subsequent structural analysis. In the present study, we demonstrate that the PISEMA and other rotating-frame pulse sequences are not suitable for the measurement of long-range heteronuclear dipolar couplings, and that they provide inaccurate values when multiple protons are coupled to a 13 C nucleus. Furthermore, we demonstrate that a laboratory-frame separated-local-field experiment is capable of overcoming these difficulties in magnetically aligned bicelles. An extension of this approach to accurately measure 13 C-31 P and 1 H-31 P couplings from phospholipids, which are useful to understand the interaction of molecules with the membrane, is also described. In these 2D experiments, natural abundance 13 C was observed from bicelles containing DMPC and DHPC lipid molecules. As a first application, these solid-state NMR approaches were utilized to probe the membrane interaction of an antidepressant molecule, desipramine, and its location in the membrane.

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Section: Introductionmentioning

confidence: 90%

Section: Two-dimensional 13 C Separated-local-field Experimentsmentioning

confidence: 99%

Section: C-31 P and 1 H-31 P Slfmentioning

confidence: 99%

Section: C-31 P and 1 H-31 P Slfmentioning

confidence: 99%

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High-Resolution 2D NMR Spectroscopy of Bicelles To Measure the Membrane Interaction of Ligands

et al. 2007

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“…The sample of Pf1 coat protein in magnetically aligned bilayers was prepared as described previously [32,33]. The phospholipids were 1,2-dimyristoyl-sn-glycero-3-phospho-choline (DMPC) and 1,2-di-O-hexyl-sn-glycero-3-phospho-choline …”

Section: Samplesmentioning

confidence: 99%

Triple resonance experiments for aligned sample solid-state NMR of 13C and 15N labeled proteins

Sinha

Grant

Park

et al. 2007

Journal of Magnetic Resonance

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Initial steps in the development of a suite of triple-resonance 1 H/ 13 C/ 15 N solid-state NMR experiments applicable to aligned samples of 13 C and 15 N labeled proteins are described. The experiments take advantage of the opportunities for 13 C detection without the need for homonuclear 13 C/ 13 C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are ~20% randomly labeled with 13 C in all backbone and side chain carbon sites and ~100% uniformly 15 N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are 13 C labeled at only the α-carbon and 15 N labeled at the amide nitrogen of a few residues. The requirement for homonuclear 13 C/ 13 C decoupling while detecting 13 C signals is avoided in the first case because of the low probability of any two 13 C nuclei being bonded to each other; in the second case, the labeled 13 C α sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the 13 C chemical shift and 1 H-13 C and 15 N-13 C heteronuclear dipolar coupling frequencies associated with the 13 Cα and 13 C′ backbone sites, which provide orientation constraints complementary to those derived from the 15 N labeled amide backbone sites. 13 C/ 13 C spin-exchange experiments identify proximate carbon sites. The ability to measure 13 C -15 N dipolar coupling frequencies and correlate 13 C and 15 N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the 13 C chemical shift and 1 H-13 C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.

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Structural genomics – expanding protein structural universe

Joachimiak

2005

Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics

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Structural Genomics has emerged as a multidisciplinary and international effort to expand the universe of protein folds by rapidly determining novel structures. This initiative takes advantage of the genome sequencing projects and technological advances in genomic analysis, molecular biology, proteomics, and structural biology. The international Structural Genomics efforts were initiated, in part, to achieve a more rapid understanding of sequence/structure relationships with the ultimate goal to make it feasible to predict an accurate 3D protein structure on the basis of sequence alone, and to elucidate novel biological function directly from primary genomic sequence. A number of Structural Genomics centers have been established to test and improve existing and develop new technologies. These centers developed the protein structure determination pipelines that include target selection, gene cloning and expression, protein and crystal production, structure determination, and structural and functional analysis. These pipelines are capable of determining hundreds of novel protein structures. Structural Genomics is rapidly expanding the universe of protein folds, creating new reagents, comprehensive libraries of expression clones, and databases for broad use. New high‐throughput technologies supported by robotics and automation in molecular biology, protein expression, purification, crystallization, and structure determination are being disseminated to the public and, therefore, contribute to the acceleration of new discoveries in biology. Ultimately, Structural Genomics will become a major component of and contributor to the next structural biology challenge – visualization of all major subcellular macromolecular assemblies of the cell.

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High-Resolution NMR Spectroscopy of Membrane Proteins in Aligned Bicelles

Cited by 104 publications

References 24 publications

High-Resolution 2D NMR Spectroscopy of Bicelles To Measure the Membrane Interaction of Ligands

High-Resolution 2D NMR Spectroscopy of Bicelles To Measure the Membrane Interaction of Ligands

Triple resonance experiments for aligned sample solid-state NMR of 13C and 15N labeled proteins

Structural genomics – expanding protein structural universe

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